HBV (hepatitis B virus, HBV) infection is a serious public health problem. According to the World Health Organization (WHO), about 20 million people have been infected with HBV within 6 billion people worldwide, of which 350 million people have chronic HBV infection; about 1 million people die each year from liver failure, cirrhosis and primary hepatocellular carcinoma (liver cancer) caused by HBV infection. Liver cancer patients worldwide, more than 75% is caused by HBV. China is an endemic area of HBV infection. The Ministry of Health of China incorporated hepatitis B vaccine into planned immunization management in 1992. According to the national hepatitis B serological surveys in 2006, after nearly 15 years of efforts, the general population in China, especially children under the age of 15, HBV infection rate has significantly decreased. Due to history background, about 93 million people are suffering from chronic HBV infection in China, including about 20 million patients with chronic hepatitis B. It is estimated that liver cirrhosis and liver cancer due to HBV cause nearly 300,000 death cases each year, wherein new hepatitis B are about 0.5 to 1 million cases. HBV disease is still an important factor that endangers people's health, hinders social development, and affects social stability for a long time. It is a serious public health problem in people-oriented society, and also a priority major health issue in China. According to the survey, the annual direct and indirect medical expense for chronic hepatitis B (including liver cirrhosis and liver cancer) in China is about 680 billion. How to effectively treat existing hepatitis B virus infection patients and hepatitis B patients has become a major problem that needs to be resolved first in the prevention and treatment of hepatitis B.
Hepatitis B vaccine immunization prevention is the most effective way to reduce the burden of disease. Gene recombination technology is the core technology of modern biotechnology; also is the mainly technology of the large-scale production of hepatitis B vaccine, and the only technology of virus-like particle hepatitis B virus surface antigen (HBsAg VLP). Saccharomyces cerevisiae was the first eukaryotic system to express foreign genes. However, there are still many shortcomings in the industrial production of S. cerevisiae expression system. For example, the exogenous gene plasmids of engineering strain are free in the cytoplasm lead to genetically unstable; the fermentation density is not enough to low production efficiency; the synthesized polypeptide chain is often hyperglycosylated. The applicant has been involved in the research and development of Hansenula polymorpha (H. polymorpha) recombinant hepatitis B vaccine since 1995. In 1998 to 2002, in Dalian Gaoxin Bio-Pharmaceutical Co., Ltd., the H. polymorpha recombinant HBsAg-adr2 hepatitis B vaccine was developed. The yield of HBsAg VLP pure stock solution was 40 mg/L, and it has been approved since 2002 in China. In 2003 to 2006, the applicant assisted Beijing Tiantan Biological Products Co., Ltd. to develop recombinant H. polymorpha HBsAg-adr2 hepatitis B vaccine. The pure stock solution of HBsAg VLP has a yield of 85 mg/liter or more; it has been submitted to the National New Drug Review in 2015, and the China patent publication number based on the vaccine is CN104232661A.
The pathogenesis of hepatitis B identified according to the prior art is as follows: After HBV infection, the HBV carriers can be generally divided into immune tolerance phase, immune clearance phase, and residual or inactive phase. The immune tolerance phase is characterized by high levels of HBV replication, positive serum HBsAg and HBeAg, high HBV DNA level (>105 copies/ml), normal alanine aminotransferase level (ALT), and no obvious abnormalities in liver histology. The immune clearance phase is characterized by serum HBV DNA level >105 copies/ml, but generally lower than the immune tolerance phase, normal or intermittently elevated aspartate aminotransferase (AST) level, and necrotic inflammation shown in liver histology. The residual or inactive phase is characterized by HBeAg-negative, anti-HBe-positive, could not be detected (PCR assay) or below the lower limit of detection, level normal, and no obvious inflammation in liver histology. However, HBV infection in adolescents and adults generally does not start from the immune tolerance phase, but initial from the immune clearance phase, which is manifested as acute hepatitis B, of which only 5%-10% develop chronic hepatitis B. However, the exact pathogenesis is still unknown.
Anti-hepatitis B virus treatment is currently the main treatment for hepatitis B virus infection and hepatitis B patients. At present, anti-hepatitis B virus drugs mainly include interferon-based immunomodulators and nucleotide analogues against HBV DNA polymerase. Although they have certain curative effects, they are not satisfactory, and most patients cannot be cured. Interferon can induce HBsAg seroclearance or seroconversion in few patients, but its high cost, need to be injected, and has certain side effects. Nucleotide analogs act on HBV DNA polymerase, which only inhibits viral replication, does not completely eliminate HBV DNA and cccDNA, and easily lead to viral resistance mutation by long-term therapy.
Therefore, in order to completely eliminate HBV and cccDNA, to develop a new and more effectively of HBV hepatitis B vaccine is urgent need. The low immune rejection of liver transplantation indicates that the human liver is an immune-tolerant organ. Liver is the target organ of hepatitis B virus (HBV) infection, so that the immune tolerance to HBV in liver is a major feature. Reversal of HBV immune tolerance is the base for the development of immunotherapy vaccine for chronic hepatitis B (CHB) patients. HBV immune tolerance is not only reflected anti-HBV immune response failed to effectively eliminate the virus in local liver, leading to persistent infection, but also reflected in the persistence of HBV, leading the systemic immune system with no response to the HBV, such as the patient in HBV immune tolerance phase is no response to HBsAg vaccine. This is also the main reason why current therapeutic vaccines are difficult to succeed in CHB patients. Liver-induced immune tolerance and its reversal mechanism will provide a theoretical basis for the development of hepatitis B vaccine.
Recent data suggest that initial CD8+T (CTL) cell activation occurs in the liver, while pre-inflammatory (i.e. innate immune activation) increases the number of survival CTLs, makes CTLs more effectively, resulting in a liver immune response to eliminate the infected HBV. In the absence of inflammation in advance (ie, innate immunity is not activated, such as infants and young children), the function of CTL is impaired and the half-life of CTL is short, resulting in liver tolerance to HBV. However, initial HBV antigen encounter immunity induced high-efficiently activated HBV CTLs in the lymph nodes outside the liver, and then into the liver, which also increases the number of survival CTLs, makes CTLs more effectively, resulting in a liver immune response to eliminate the infected HBV. This immune mechanism for the two sites provides a theoretical basis for the development of subcutaneous and intramuscular injection of hepatitis B vaccine, which leads to liver immune response and HBV clearance.
According to the journal “A Whole Recombinant Yeast-Based Therapeutic Vaccine that is comprised of heat-inactivated, whole recombinant Saccharomyces cerevisiae yeast cells expressing disease-related antigens” of Thomas H. King (US) in 2014, which relates to a therapeutic vaccine based on a heat-inactivated, whole recombinant Saccharomyces cerevisiae yeast cells expressing disease-related antigen. The study pioneered a therapeutic vaccine platform that uses intracellular recombinantly expressed proteins as antigens and heat-inactivated whole Saccharomyces cerevisiae cells as adjuvant. In addition, the HBV antigen expressed by a hepatitis B therapeutic vaccine (which has been numbered as GS4774 under the platform) is an x-s-core antigen as fusion protein. This yeast vector provides multiple antigens into the MHC class I and class II antigen presentation pathways, stimulates potent CD4+ and CD8+ cell responses, and disrupts antigen tolerance in immunogenic mouse model. The yeast vector is also not easily being neutralized in body, and is therefore suitable for repeated administration to obtain long-term immunological stress, ideally eliminating chronic intracellular infections such as HCV and HBV.
According to the journal of Huang in 2010, β-Glucan particles (GPs) which are purified from Saccharomyces cerevisiae cell walls have >85% β1,3-d-glucan polymers, ˜2% chitin, and <1% lipids and protein, with the rest being mostly ash and moisture. In in vitro T-cell proliferation assays, ovalbumin (OVA) was complexed into the hollow GP shells (GP-OVA) to as vaccine, and free OVA as control antigen. At concentrations from 0.03 μg/ml to 0.5 μg/ml, GP-OVA stimulated OT-I and OT-II T-cell proliferation. In contrast, free OVA failed to stimulate proliferation of either OT-I or OT-II T cells. In order to achieve similar stimulation effects of GP-OVA, 100 times or higher concentrations of free OVA were required. These results demonstrate that: (1) Virus-like particle GPS is an efficiently t agonist of the Dectin-1 receptor. (2) Compared with free OVA, antigens delivered in Virus-like particle GP-OVA were more efficiently processed and presented by DCs (dendritic cells).
In 2003 to 2005, American scientists reported the results of a series of studies on hepatitis B virus infection in chimpanzees. The mechanism controlling disease is the covalently closed circular DNA (cccDNA) of the hepatocyte nuclear HBV pool. The HBV-specific CD8+T (CTL cells), which produce INF-γ, massively influx into liver and target to hepatocytes infected with HBV, and the cccDNA clearance and hepatocytes infected HBV reversion are related to the INF-γ produced by liver CD8+ T cells. These results suggest that cccDNA clearance is a two-step process mediated by cellular immune responses: In the first step is to reduce the pool of cccDNA molecules by more than 90% without cell damage, thereby eliminating the precursor of HBV-relaxed circular deoxyribonucleic acid, and the second step is to improve the process of destroying infected liver cells and trigger an immune reversion.
In 2014, Dr. Zeng Zhutian of the University of Science and Technology of China reported that, by hydrodynamic injection of HBV persistent mouse mimic the immune tolerance phase of chronically infected HBV patients, the combination therapy of IL-12 pretreatment with IL-12 and HBsAg VLP vaccine, which is called IL-12-based vaccine therapy, can effectively reverse HBV systemic immune tolerance, and lead to HBV clearance. The levels of follicular-like helper T cells (Tfh, which are in lymph node) and germinal center B cells (GC B) of HBV mice were significantly increased after undergoing IL-12-based vaccine therapy. Correspondingly, HBsAg-specific IgG-producing cells in spleen cells were also significantly increased, and most of the mice showed protective antibody anti-HBs in the serum of the late treatment. In addition, the ability of T cells to stimulate HBsAg stimulation in vitro was also significantly restored after IL-12 combination vaccine treatment.
The China patent publication number CN102038948A described that hepatitis B vaccine immunized healthy mice with whole recombinant H. polymorpha cells expressing HBsAg VLP antigen, and achieved well results at the cellular and protein molecular levels: induction of the activation, maturation and proliferation of high-level dendritic cells (DCs), induction of extremely high-level of anti-HBs immune response, Induction of higher level of IFN-γ secretion, and result in a significant HBsAg-specific CTL (cytotoxic T lymphocyte) response. It guides the development of chronic hepatitis B vaccine. The test mice were immunized with high doses: 2×108 recombinant H. polymorpha cells and 2 μg hepatitis B surface antigens (about 20 times the half effective dose of the ED50 test in mice) it expressed was the key that obtained good results. However, there is a safety limit for the number of recombinant H. polymorpha cells used for human injection (8−12×108 recombinant H. polymorpha cells). The expression level of HBsAg VLP antigen in recombinant H. polymorpha cells disclosed in is too low, when intended for human injection which a single injection of low dose of HBsAg, is difficult to exert a therapeutic effect of this vaccine. Moreover, CN102038948A does not mention any HBsAg-specific CTL epitopes and heat-inactivation processes. Therefore, providing recombinant H. polymorpha cells having a high expression level of HBsAg VLP antigen and preferably a HBsAg-specific CTL epitope and a heat inactivation process, so as to provide a hepatitis B therapeutic vaccine based on inactivated whole recombinant H. polymorpha cell expressing HBsAg has become an important issue in the art.